It is well known that development and function of the hematopoietic system is supported and regulated by its microenvironment of stromal origin (post-natally, hematopoiesis is totally restricted to skeletal bones) [Dorshkind, K (1990) Ann Rev Immunol 8:111; Bentley, S. A. (1981) Exp Hematol 9:303; Smith, B. R. (1990) Yale J. Biol Med 63:371-380].
Normal bone marrow function depends on co-existence of normal hematopoietic stem cells and its microenvironment, mostly located in the medullary part of the bones. Stem cells cannot function properly in the absence of an adequate microenvironment. Whereas in malignant and non-malignant hematologic diseases caused by deficiency or abnormal stem cells, stem cell transplantation is the treatment of choice that results in cure, in diseases such as myelodysplastic syndrome (MDS), myelofibrosis and other conditions associated with abnormal microenvironment, pancytopenia may occur despite presence of apparently normal hematopoietic cells with no recognizable cytogenetic abnormality. Indeed, in such patients, correction of refractory anemia may sometimes be accomplished following autologous bone marrow transplantation, although the success rate is rather poor in comparison with the outcome of transplants in other hematologic malignancies. In MDS with excess blasts (also known as refractory anemia with excess blasts, RAEB), with blastic transformation (RAEB-T), or with fully overt leukemia, the disease is usually caused by a combination of malignant marrow stem cells and abnormal marrow microenvironment. The success rate in terms of engraftment and disease-free survival in patients with MDS is lower than that observed in patients with acute and chronic leukemia, most likely due to the fact that the replacement of stem cells alone may not be sufficient for a proper engraftment and normal bone marrow function. Theoretically, one could improve the outcome of bone marrow transplantation (BMT) by both replacing abnormal stem cells and providing new microenvironment.
Allogeneic BMT involves the transfer of allogeneic marrow stem cells from a healthy donor to a patient in need. Following BMT, the patient's bones and hematopoietic niches are reconstituted with donor cells, and the entire hematopoietic system including red blood cells, platelets, nucleated cells, the circulating and tissue-bound reticuloendothelial system and the entire immune system, are converted to be of donor origin [Slavin S. and Nagler A. (1991) Curr Opin Oncol 3(2):254-71]. BMT is widely applicable for treating and correcting a large number of hematopoietic disorders, including a deficiency in any of the bone marrow products, as well as in a need for replacement of abnormal stem cells and in a large number of genetic disorders [Buckley R. H. et al. (1993) Semin Hematol 30(4, Suppl 4):92-101; Brochstein J. A. (1992) Oncology (Huntingt) 6(3):51-8; discussion 58, 63-6]. Alternatively, BMT is widely used for cancer therapy and for rescue of patients receiving myeloablative chemoradiotherapy [Slavin S. and Nagler A. (1991) id ibid]. There are many additional potential applications for BMT, such as induction of transplantation tolerance in organ transplantation [Sykes M. and Sacks D. H. (1990) Semin Immunol 2:401-417], and as a platform for immunotherapy associated with donor lymphocyte infusions [Champlin R. (1991) Transplant Proc 23(4):2123-7; Brenner M. K. and Heslop H. E. (1991) Baillieres Clin Haematol 4(3):727-49; Slavin S. et al. (1991) Baillieres Clin Haematol 4(3):715-25]. Following myeloablative conditioning, BMT can be used for replacing all host immunohematopoietic system with donor type cells. Alternatively, following non-myeloablative conditioning, the role of BMT could be directed to achieving durable engraftment of donor hematopoiesis side by side with host hematopoiesis for induction of mixed chimerism.
Unfortunately, several limitations restrict the applicability of BMT, such as:    1. Certain hematological diseases for which BMT is indicated may also affect the microenvironment of the marrow and, therefore, stem cell transplantation alone may not be sufficient for complete hematological recovery [Enright H. and McGlave P. B. (1995): Curr Opin Hematol 2(4):293-9; Koijima S. (1998) Int J Hematol 68(1):19-28; Gidali J. et al. (1996) Stem Cells 14(5):577-583].    2. Efficient consistent engraftment of allogeneic bone marrow cells (BMC), especially purified stem cells or T cell-depleted stem cells, requires transfer of a large number of stem cells which may be difficult to obtain or are even unavailable (e.g. cord blood stem cells, with limited number of cells; child to adult transplant; etc.) [Reisner Y. and Martelli M. F. (1995) Immunol Today 16:437; Rowe J. M. et al. (1994) Ann Intern Med. 120(2):143-58].    3. Stem cell engraftment in patients with hematopoietic disorders, especially in patients who are treated following mandatory conditioning with irradiation or chemotherapy, is considerably less efficient because of the damaged hematopoietic microenvironment [Enright H. and McGlave P. B. (1995) id ibid.; O'Flaherty E. et al. (1995) Bone Marrow Transplant 15:207].    4. Allogeneic BMT as applied to date in patients with hematological disorders is frequently complicated by procedure-related toxicity, poor engraftment and anti-host reaction leading to graft-vs-host disease (GVHD) [Lazarus H. M. et al. (1997) Bone Marrow tranplant 19:577; Quinones R. R. (1993) American J. Pediatric Hematology/Oncology 15(1):3-17].
In view of the above limitations, one of the main objects of the present invention is the improved engraftmient of BMC in recipients with damaged hematopoietic microenvironment. As the inventors have recently shown, transplantation of BMC within donor hematopoietic microenvironment lowered incidence of GVHD [Gurevitch O. A. et al. (1999) Transplantation 68:1362-1368]. Therefore, when allogeneic BMT is associated with procedure-related toxicity due to use of immunosuppressants for treatment of GVHD, complications could be avoided. Furthermore, improvement in transplantation procedure resulted in reduction of the number of BMC required for transplantation.
The present inventors have previously shown that induced bones and hematopoietic microenvironment, developed in mice after ectopic implantation of demineralized bone or tooth matrix, reveal the main properties of skeletal bones [Gurevitch O. A. (1990) Int. J. Cell. Clon 8:130-137; Gurevitch O. A. and Fabian I. (1993) Stem Cells 11:56-61]. The following observations were made:                a. The hematopoietic microenvironment in induced bones is capable of supporting fully developed three-lineage hematopoiesis.        b. The hematopoietic microenvironment in induced bones is capable of maintaining the proliferative potential of hematopoietic stem cells as could be best documented by colony forming units in the spleen (CFU-S).        c. De novo induced hematopoietic microenvironment is capable of long-term maintenance, remodeling and self-renewal without additional application of any exogenous supplement. Once developed, induced bone becomes a permanent structure in ectopic locations with a functionally active hematopoietic niche.        
These observations suggest that induction of hematopoietic microenvironment could be a promising approach for correcting stromal and hematopoietic disorders as well as for enhancing hematopoiesis.
Engraftment of transplanted BMC is improved when hematopoietic cells are transferred together with a bone transplant [Ishida T. et al. (1994) J Immunol 152:3119-3127; Hisha H. et al. (1995) Exp Hematol 23:347-352; Sandhu J. S. et al. (1996) Blood 88(6):1973-1982; Nakagawa T. et al. (1993) Arthritis and Rheumatism 36:263-268] or injection of stromal cells of the donor origin [Lazarus, H. M. et al. (1995) Bone Marrow Transplantation 16:557-564; El-Badri, N. S. et al. (1998) Exp Hematol 26:110-116]. In 1999, the present inventors have shown that GVHD is suppressed if hematopoietic BMC are transferred within their own hematopoietic microenvironment [Gurevitch O. A. (1999) id ibid.]. Allogeneic bone marrow was transplanted not in the usual form of a single cell suspension, where only hematopoietic stem cells are capable of engraftment, but in the form of an undisturbed bone marrow plug evacuated from the femural marrow cavity of donor mouse. The donor bone marrow plug was placed under the kidney capsule of the recipient. In this case, which is an almost not clinically applicable, mesenchymal cell present in the transplant in the form of undisturbed multi-cellular structures produce ectopic (extraskeletal) microenvironment for hematopoietic cells transplanted within the same bone marrow plug.
However, transplantation of bone osteoblasts or bone marrow plugs has not yet been widely accepted in clinical practice because of many technical limitations.
A major shortcoming for all current BMC transplantation procedures is their inability to simultaneously transfer both hematopoietic cells and stromal microenvironment supporting hematopoiesis. In BMC transplantation procedures currently used in hematological practice, in fact only hematopoietic cells from donor are engrafted in the recipient.
Based on the notion that BMC may provide a source for both hematopoietic cells and mesenchymal stem cells capable of induced osteogenesis, the inventors propose transplantation of a composition comprising BMC and DBM and/or MBM (materials stimulating and supporting osteogenic development of mesenchymal precursor cells), allowing for simultaneous development of hematopoietic and stromal tissues originating from donor BMC transplant.
More specifically, the composition of the present invention (comprising BMC and DBM and/or MBM) is transplanted directly into a bone marrow cavity of the recipient or extraskeletally, resulting in de novo formation of stromal microenvironment, which will support hematopoiesis, and hematopoietic tissue from the same transplanted BMC suspension.
Therefore, it is a major object of the present invention a mixture of bone marrow cells with demineralized or mineralized bone matrix, for use for transplantation into patients in need of such treatment, as for example patients suffering from a hematological disorder.
These and other objects of the present invention will become apparent as the description proceeds.